Active and inactive CC-chemokine receptor

ABSTRACT

A peptide has an amino acid sequence having more than 80% homology with the amino acid sequence listed as SEQ ID NO:4. A nucleic acid molecule has more than 80% homology with one of the nucleic acid sequences listed as SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:3. Ligands, anti-ligands, cells vectors relating to the peptide and/or nucleic acid molecule are also used.

This is a continuation application under 37 CFR 1.53 OF U.S. patentapplication Ser. No. 08/810,028 filed Mar. 3, 1997 now abandoned.

BACKGROUND

1. Field of the Invention

The present invention concerns new peptides and the nucleic acidmolecules encoding said peptides, the vector comprising said nucleicacid molecules, the cells transformed by said vector, inhibitorsdirected against said peptides or said nucleic acid molecules, apharmaceutical composition and a diagnostic and/or dosage devicecomprising said products, and non human transgenic animals expressingthe peptides according to the invention or the nucleic acid moleculesencoding said peptides.

The invention further provides a method for determining ligand binding,detecting expression, screening for drugs binding specifically to saidpeptides and treatments involving the peptides or the nucleic acidmolecules according to the invention.

2. Technological Background of the Art

Chemotactic cytokines, or chemokines, are small signalling proteins thatcan be divided in two subfamilies (CC- and CXC-chemokines) depending onthe relative position of the first two conserved cysteines. Interleukin8 (IL-8) is the most studied of these proteins, but a large number ofchemokines (Regulated on Activation Normal T-cell Expressed and Secreted(RANTES), Monocyte Chemoattractant Protein 1 (MCP-1), MonocyteChemoattractant Protein 2 (MCP-2), Monocyte Chemoattractant Protein 3(MCP-3), Growth-Related gene product a (GROα), Growth-Related geneproduct β (GRO)β, Growth-Related gene product γ(GROγ), MacrophageInflammatory Protein 1 α(MIP-1α) and β, etc.) has now been described[4]. Chemokines play fundamental roles in the physiology of acute andchronic inflammatory processes as well as in the pathologicaldysregulations of these processes, by attracting and simulating specificsubsets of leucocytes [32]. RANTES for example is a chemoattractant formonocytes, memory T-cells and eosinophils, and induces the release ofhistamine by basophils. MCP-1, released by smooth muscle cells inarteriosclerotic lesions, is considered as the factor (or one of thefactors) responsible for macrophage attraction and, therefore, for theprogressive aggravation of the lesions [4].

MIP-1α, MIP-1βand RANTES chemokines have recently been described asmajor HIV-suppressive factors produced by CD8⁺ T-cells [9].CC-chemokines are also involved in the regulation of human myeloidprogenetor cell proliferation [6, 7].

Recent studies have demonstrated that the actions of CC- andCXC-chemokines are mediated by subfamies of G protein-coupled receptors.To date, despite the numerous functions attributed to chemokines and theincreasing number of biologically active ligands, only six functionalreceptors have been identified in human. Two receptors for interleukin-8(IL-8) have been described [20, 29]. One (IL-8RA) binds IL-8specifically, while the other (IL-8RB) binds IL-8 and otherCXC-chemokines, like GRO. Among receptors binding CC-chemokines, areceptor, designated CC-chemokine receptor 1 (CCR1), binds both RANTESand MIP-1α [31], and the CC-chemokine receptor 2 (CCR2) binds MCP-1 andMCP-3 [8, 44, 15]. Two additional CC-chemokine receptors were clonedrecently: the CC-chemokine receptor 3 (CCR3) was found to be activatedby RANTES, MIP-1α and MIP-1β [10]; the CC-chemokine receptor 4 (CCR4)responds to MIP-1, RANTES and MCP-1 [37]. In addition to these sixfunctional receptors, a number of orphan receptors have been cloned fromhuman and other species, that are structurally related to either CC- orCXC-chemokine receptors. These include the human BLR1 [13], EBI1 [5],LCR1 [21], the mouse MIP-1 RL1 and MIP-1 RL2 [17] and the bovine PPR1[25]. Their respective ligand(s) and function(s) are unknown at present.

SUMMARY OF THE INVENTION

The present invention is related to a peptide having at least an aminoacid sequence which presents more than 80%, advantageously more than90%, preferably more than 95%, homology with the amino acid sequence asrepresented in SEQ ID NO:4.

Preferably, said peptide has also at least an amino acid sequence whichpresents more than 80%, advantageously more than 90%, preferably morethan 95%, homology with the amino acid sequence as represented in SEQ IDNO:5.

According to another embodiment of the present invention, the peptidehas at least an amino acid sequence which presents more than 80%,advantageously more than 90%, preferably more than 95%, homology withthe amino acid sequence as represented in SEQ ID NO:6.

The present invention is also related to the amino acid sequence of SEQID NO:4, SEQ ID NO:5, SEQ ID NO:6 or a portion thereof (represented inthe FIG. 1).

A “portion of an amino acid sequence” means one or more amino acidssegments having the same or improved binding properties of the wholepeptide according to the invention. Said portion could be an epitopewhich is specifically binded by a ligand of the peptide which could be aknown “natural ligand” of said peptide, an agonist or an analog of saidligand, or an inhibitor capable of competitively inhibiting the bindingof said ligand to the peptide (including the antagonists of said ligandto the peptide).

Specific examples of said portions of amino acid sequence and theirpreparation process are described in the publication of Rucker J. et al.(Cell, Vol. 87, pp. 437-446 (1996)) incorporated herein by reference.

According to the invention, said portion of the amino acid sequence ofthe peptide according to the invention comprises the N-terminus segmentand the first extracellular loop of the peptide.

Therefore, according to the invention, the amino acid sequence asrepresented in SEQ ID NO:4 is the common amino acid sequence of SEQ IDNO:5 and of SEQ ID NO:6 (see also FIG. 1). Therefore, a first industrialapplication of said amino acid sequence is the identification of thehomology between said amino acid sequence and the screening of variousmutants encoding a different amino acid sequence than the one previouslydescribed, and the identification of various types of patient which maypresent a predisposition or a resistance to the disorders described inthe following specification.

Preferably, the peptide according to the invention or a portion thereofis an active CC-chemokine receptor.

Advantageously, the CC-chemokine receptor according to the invention isstimulated by the MTI1β chemokine at a concentration less or equal to 10nm, and is advantageously also stimulated by the MIP-1α or RANTESchemokines. However, said chemokine receptor is not stimulated by theMCP-1, MCP-2, MCP-3, IL-8 and GROα chemokines.

In addition, the peptide according to the invention or a portion thereofis also a receptor of HIV viruses or a portion of said HIV viruses.

It is meant by “HIV viruses”, HIV-1 or HIV-2 and all the various strainsof HIV viruses which are involved in the development of AIDS. It ismeant by a “a portion of HIV viruses”, any epitope of said viruses whichis able to interact specifically with said receptor. Among said portionsof viruses which may be involved in the interaction with the peptideaccording to the invention, are peptides encoded by the ENV and GAGviruses genes.

Preferably, said portion of HRV viruses is the glycopeptide gp120/160(membrane-bound gp160 or the free gp derived therefrom) or a portionthereof.

It is meant by a “portion of the glycopeptide gp120/160” any epitope,preferably an immuno-dominant epitope, of said glycopeptide which mayinteract specifically with the peptide according to the invention, suchas for instance the V3 loop (third hypervariable domain).

According to another embodiment of the present invention, the peptideaccording to the invention is an inactive CC-chemokine receptor. Anexample of such inactive CC-chemokine receptor is encoded by the aminoacid sequence as represented in SEQ ID NO:5.

It is meant by an “inactive CC-chemokine receptor” a receptor which isnot stimulated by any known CC-chemokine, especially the MIP-1β, MIP-1αor RANTES chemokines.

The peptide represented in SEQ ID NO:6 according to the invention is aninactive receptor which is not a receptor of HIV viruses or of a portionof said HIV viruses, which means that said inactive receptor does notallow the entry of said HIV viruses into a cell which presents at itssurface said inactive receptor.

Advantageously, the peptide according to the invention is a humanreceptor.

The present invention concerns also the nucleic acid molecule havingmore than 80%, preferably more than 90%, homology with one of thenucleic acid sequences of SEQ ID NO. 1, SEQ ID NO: 2 and SEQ ID NO. 3shown in FIG. 1.

Preferably, said nucleic acid molecule has at least the nucleic acidsequence shown in SEQ ID NO. 1, SEQ ID NO. 2 or SEQ ID NO. 3 of FIG. 1or a portion thereof.

It is meant by a “portion of said nucleic acid molecule” any nucleicacid sequence of more than 15 nucleotides which could be used in orderto detect and/or reconstitute said nucleic acid molecule or itscomplementary strand. Such portion could be a probe or a primer whichcould be used in genetic amplification using the PCR, LCR, NASBA or CPRtechniques for instance.

The present invention concerns more specifically the nucleic acidmolecules encoding the peptide according to the invention. Said nucleicacid molecules are RNA or DNA molecules such as a cDNA molecule or agenomic DNA molecule.

The present invention is also related to a vector comprising the nucleicacid molecule according to the invention. Preferably, said vector isadapted for expression in a cell and comprises the regulatory elementsnecessary for expressing the amino acid molecule in said celloperatively linked to the nucleic acid sequence according to theinvention as to permit expression thereof.

Preferably, said cell is chosen among the group consisting of bacterialcells, yeast cells, insect cells or mammalian cells. The vectoraccording to the invention is a plasmid, preferably a pcDNA3 plasmid, ora virus, preferably a baculovirus, an adenovirus or a semliki forestvirus.

The present invention concerns also the cell, preferably a mammaliancell, such as a CHO-K1 or a HEK293 cell, transformed by the vectoraccording to the invention. Advantageously, said cell is non neuronal inorigin and is chosen among the group consisting of CHO-K1, HEK293,BHK21, COS-7 cells.

The present invention also concerns the cell (preferably a mammaliancell such as a CHO-K1 cell) transformed by the vector according to theinvention and by another vector encoding a protein enhancing thefunctional response in said cell. Advantageously, said protein is theGα15 or Gα16 (G protein, α subunit). Advantageously, said cell is thecell CHO-K1-pEFIN hCCR5-1/16.

The present invention is also related to a nucleic acid probe comprisinga nucleic acid molecule of at least 15 nucleotides capable ofspecifically hybridizing with a unique sequence included within thesequence of the nucleic acid molecule according to the invention, Saidnucleic acid probe may be a DNA or a RNA.

The invention concerns also an antisense oligonucleotide having asequence capable of specifically hybridizing to an mRNA moleculeencoding the peptide according to the invention so as to preventtranslation of said mRNA molecule or an antisense oligonucleotide havinga sequence capable of specifically hybridizing to the cDNA moleculeencoding the peptide according to the invention.

Said antisense oligonucleotide may comprise chemical analogs ofnucleotide or substances which inactivate mRNA, or be included in an RNAmolecule endowed with ribozyme activity.

Another aspect of the present invention concerns a ligand or ananti-ligand (preferably an antibody) other than known “natural ligands”,which are chosen among the group consisting of the MIP-1β, MIP-1α orRANTES chemokines, HIV viruses or a portion of said HIV viruses, whereinsaid ligand is capable of binding to the receptor according to theinvention and wherein said anti-ligand is capable of (preferablycompetitively) inhibiting the binding of said known “natural ligand” orthe ligand according to the invention to the peptide according to theinvention.

The exclusion in the above identified definition of known chemokines,HIV viruses or a portion of said HIV viruses, does not include variantsof said “natural” viruses or said “natural” portion which may beobtained for instance by genetic engineering and which may mimic theinteraction of said viruses and portion of said viruses to the peptideaccording to the invention.

Advantageously, said antibody is a monoclonal antibody which ispreferably directed to an epitope of the peptide according to theinvention and present on the surface of a cell expressing said peptide.

Preferably, said antibody is produced by the hybridome cellAchCCR5-SAB1A7.

The invention concerns also the pharmaceutical composition comprisingeither an effective amount of the peptide according to the invention (inorder to delude the HIV virus from the natural peptide present at thesurface of a mammalian cell and stop the infection of said mammaliancell by the HIV virus), or an effective amount of the above identifieddescribed ligand and/or anti-ligand, or An effective amount ofoligonucleotide according to the invention, effective to decrease theactivity of said peptide by passing through a cell membrane and bindingspecifically with MRNA encoding the peptide according to the inventionin the cell so as to prevent it translation. The pharmaceuticalcomposition comprises also a pharmaceutically acceptable carrier,preferably capable of passing through said cell membrane.

Preferably, in said pharmaceutical composition, the oligonucleotide iscoupled to a substance, such as a ribozyme, which inactivates mRNAencoding the peptide according to the invention.

Preferably, the pharmaceutically acceptable carrier comprises astructure which binds to a receptor on a cell capable of being taken upby cell after binding to the structure. The structure of thepharmaceutically acceptable carrier in said pharmaceutical compositionis capable of binding to a receptor which is specific for a selectedcell type.

The present invention concerns also a transgenic non human mammaloverexpressing (or expressing ectopically) the nucleic acid moleculeencoding the peptide according to the invention, The present inventionalso concerns a transgenic non human mammal comprising an homologousrecombination knockout of the native peptide according to the invention.

According to a preferred embodiment of the invention, the transgenic nonhuman mammal whose genome comprises antisense nucleic acid complementaryto the nucleic acid according to the invention is so placed as to betranscripted into antisense MRNA which is complementary to the MRNAencoding the peptide according to the invention and which hybridizes toMRNA encoding said peptide, thereby reducing its translation.Preferably, the transgenic non human mammal according to the inventioncomprises a nucleic acid molecule encoding the peptide according to theinvention and comprises additionally an inducible promoter or a tissuespecific regulatory element.

Preferably, the transgenic non human mammal is a mouse.

The invention relates to a method for determining whether a ligand canbe specifically bound to the peptide according to the invention, whichcomprises contacting a cell transfected with a vector expressing thenucleic acid molecule encoding said peptide with the ligand underconditions permitting binding of ligand to such peptide and detectingthe presence of any such ligand bound specifically to said peptide,thereby determining whether the ligand binds specifically to saidpeptide.

The invention relates to a method for determining whether a ligand canspecifically bind to a peptide according to the invention, whichcomprises preparing a cell extract from cells transfected with a vectorexpressing the nucleic acid molecule encoding said peptide, isolating amembrane fraction from the cell extract, contacting the ligand with themembrane fraction under conditions permitting binding of the ligand tosuch peptide and detecting the presence of any ligand bound to saidpeptide, thereby determining whether the compound is capable ofspecifically binding to said peptide. Preferably, said method is usedwhen the ligand is not previously known.

The invention relates to a method for determining whether a ligand is anagonist of the peptide according to the invention, which comprisescontacting a cell transfected with a vector expressing the nucleic acidmolecule encoding said peptide with the ligand under conditionspermitting the activation of a functional peptide response from the celland detecting by means of a bio-assay, such as a modification in asecond messenger concentration (preferably calcium ions or inositolphosphates such as IP₃) or a modification in the cellular metabolism(preferably determined by the acidification rate of the culture medium),an increase in the peptide activity, thereby determining whether theligand is a peptide agonist.

The invention relates to a method for determining whether a ligand is anagonist of the peptide according to the invention, which. comprisespreparing a cell extract from cells transfected with a vector expressingthe nucleic acid molecule encoding said peptide, isolating a membranefraction from the cell extract, contacting the membrane fraction withthe ligand under conditions permitting the activation of a functionalpeptide response and detecting by means of a bio-assay, such as amodification in the production of a second messenger (preferablyinositol phosphates such as IP₃), an increase in the peptide activity,thereby determining whether the ligand is a peptide agonist.

The present invention relates to a method for determining whether aligand is an antagonist of the peptide according to the invention, whichcomprises contacting a cell transfected with a vector expressing thenucleic acid molecule encoding said peptide with the ligand in thepresence of a known peptide agonist, under conditions permitting theactivation of a functional peptide response and detecting by means of abio-assay, such as a modification in second messenger concentration(preferably calcium ions or inositol phosphates such as IP₃) or amodification in the cellular metabolism (preferably determined by theacidification rate of the culture medium), a decrease in the peptideactivity, thereby determining whether the ligand is a peptideantagonist.

The present invention relates to a method for determining whether aligand is an antagonist of the peptide according to the invention, whichcomprises preparing a cell extract from cells transfected with anexpressing the nucleic acid molecule encoding said peptide, isolating amembrane fraction from the cells extract, contacting the membranefraction with the ligand in the presence of a known peptide agonist,under conditions permitting the activation of a functional peptideresponse and detecting by means of a bio-assay, such as a modificationin the production of a second messenger, a decrease in the peptideactivity, thereby determining whether the ligand is a peptideantagonist.

Preferably, the second messenger assay comprises measurement of calciumions or inositol phosphates such as IP₃.

Preferably, the cell used in said method is a mammalian cell nonneuronal in origin, such as CHO-K1, HEK293, BHK21, COS-7 cells.

In said method, the ligand is not previously known.

The invention is also related to the ligand isolated and detected by anyof the preceding methods.

The present invention concerns also the pharmaceutical composition whichcomprises an effective amount of an agonist or an antagonist of thepeptide according to the invention, effective to reduce the activity ofsaid peptide and a pharmaceutically acceptable carrier.

It is meant by “an agonist or an antagonist of the peptide according tothe invention”, all the agonists or antagonists of the known “naturalligand” of the peptide as above described.

Therefore, the previously described methods may be used for thescreening of drugs to identify drugs which specifically bind to thepeptide according to the invention.

The invention is also related to the drugs isolated and detected by anyof these methods.

The present invention concerns also a pharmaceutical compositioncomprising said drugs and a pharmaceutically acceptable carrier.

The invention is also related to a method of detecting expression of apeptide according to the invention by detecting the presence of MRNAcoding for a peptide, which comprises obtaining total RNA or total MRNAfrom the cell and contacting the RNA or MRNA so obtained with thenucleic acid probe according to the invention under hybridizingconditions and detecting the presence of MRNA hybridized to the probe,thereby detecting the expression of the peptide by the cell.

Said hybridization conditions are stringent conditions.

The present invention concerns also the use of the pharmaceuticalcomposition according to the invention for the treatment and/orprevention of inflammatory diseases, including rheumatoid arthritis,glomerulonephritis, asthma, idiopathic puhnonary fibrosis and psoriasis,viral infections including Human Immunodeficiency Viruses 1 and 2 (HIV-1and 2), cancer including leukaemia, atherosclerosis and/or auto-immunedisorders.

The present invention concerns also a method for diagnosing apredisposition or a resistance to a disorder associated with theactivity of the peptide according to the invention and/or associatedwith infectious agents such as HIV viruses in a subject. Said methodcomprises:

a) obtaining nucleic acid molecules encoding the peptide according tothe invention from the cells of the subject;

b) possibly performing a restriction digest of said nucleic acidmolecules with a panel of restriction enzymes;

c) possibly electrophoretically separating the resulting nucleic acidfragments on a sized gel;

d) contacting the resulting gel or the obtained nucleic acid moleculewith a nucleic acid probe labelled with a detectable marker and capableof specifically hybridizing to said nucleic acid molecule (saidhybridization being made in stringent hybridization conditions);

e) detecting labelled bands or the in situ nucleic acid molecules whichhave hybridized to the said nucleic acid molecule labelled with adetectable marker to create a unique band pattern or an in situ markingspecific to the subject;

f) preparing other nucleic acid molecules encoding the peptide accordingto the invention obtained from the cells of other patients for diagnosisby step a-e; and

g) comparing the unique band pattern specific to the nucleic acidmolecule of subjects suffering from the disorder from step e and thenucleic acid molecule obtained for diagnosis from step f to determinewhether the patterns are the same or different and to diagnose thereby apredisposition or a resistance to the disorder if the patterns are thesame or different.

The present invention is also related to a method for diagnosing apredisposition or a resistance to a disorder associated with theactivity of a specific allele of the peptide according to the inventionor the presence of said peptide at the surface of cells and/orassociated with infectious agents such as HIV viruses present in asubject. Said method comprises:

a) obtaining a sample of a body fluid, preferably a blood samplecomprisingantigen presenting cells, from a subject;

b) adding to said sample a ligand and/or an anti-ligand according to theinvention;

c) detecting the cross-reaction between said ligand and/or saidanti-ligand and the specific peptide according to the invention; and

d) determining whether the peptide corresponds to a receptor or aninactive receptor according to the invention and diagnosing thereby apredisposition or a resistance to the disorder according to the type ofthe peptide present in the body fluid of the subject.

The present invention concerns also a diagnostic and/or dosage device,preferably a kit, comprising the peptides, the nucleic acid molecules,the nucleic acid probes, the ligands and/or the anti-ligands accordingto the invention, their portions (such as primers, probes, epitopes, . .. ) or a mixture thereof, being possibly labelled with a detectablemarker.

Said diagnostic and/or dosage device comprises also the reactants forthe detection and/or the dosage of antigens, antibodies or nucleic acidsequences through a method selected from the group consisting of in situhybridization, hybridization or recognition by marked specificantibodies, specially ELISA® (Enzyme Linked immunosorbent Assay) or RIA®(Radio Immunoassay), methods on filter, on a solid support, in solution,in “sandwich”, on gel, by Dot blot hybridization, by Northern blothybridization, by Southern blot hybridization, by isotopic ornon-isotopic labelling (such as immunofluorescence or biotinylation), bya technique of cold probes, by genetic amplification, particularly PCR,LCR, NASBA or CPR, by a double immunodiffusion, by acounter-immunoelectrophoresis, by haemagglutination and/or a mixturethereof.

A last aspect of the present invention concerns a method of preparingpeptides according to the invention, which comprises:

a) constructing a vector adapted for expression in a cell whichcomprises the regulatory elements necessary for the expression ofnucleic acid molecules in the cell operatively linked to nucleic acidmolecule encoding said peptide so as to permit expression thereof,wherein the cell is preferably selected from the group consisting ofbacterial cells, yeast cells, insect cells and mammalian cells;

b) inserting the vector of step a in a suitable host cell;

c) incubating the cell of step b under conditions allowing theexpression of the peptide according to the invention;

d) recovering the peptide so obtained; and

e) purifying the peptide so recovered, thereby preparing an isolatedpeptide according to the invention.

The deposits of micro-organisms AchCCR5-SAB1A7 and CHO-K1-PEFINHCCR5-1/16 were made according to the Budapest Treaty in the BelgiumCoordinated Collection of Micro-organisms (BCCM), Laboratorium voorMoleculaire Biologie (LMBP), Universiteit Gent, K. L. Ledeganckstraat35, B-9000 GENT, BELGIUM.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 represents the primary structure of the peptides according to theinvention.

FIG. 2 represents the amino acids sequence of the active human CCR5chemokine receptor according to the invention aligned with that of thehuman CCR1, CCR2b, CCR3 and CCR4 receptors. Amino acids identical withthe active CCR5 sequence are boxed.

FIG. 3 shows the chromosomal organisation of the human CCR2 and CCR5chemokine receptor genes.

FIG. 4 shows the functional expression of the human active CCR5 receptorin a CHO-K1 cell line.

FIG. 5 represents the distribution of MRNA encoding the CCR5 receptor ina panel of human cell lines of haematopoietic origin.

FIG. 6 represents the structure of the mutant form of human CCR5receptor.

FIG. 7 represents the quantification of ENV proteins-mediated fusion byluciferase assays.

FIG. 8 represents genotyping of individuals by PCR and segregation ofthe CCR5 alleles in CEPH families.

FIG. 9 represents the FACS analysis of sera anti-CCR5 on a CCR5-CHO cellline according to the invention.

FIG. 10 represents the inhibition of HIV infectivity with anti-CCR5antibodies.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

1. Experimentals

Materials

Recombinant human chemoklnes, including MCP-1, MIP-1α, MIP-1β, RANTES,IL-8 and GROα were obtained from R & D Systems (London, UK).[¹²⁵I]MIP-1α (specific activity, 2200 Ci/mmol) was obtained from DupontNEN (Brussels, Belgium). Chemokines obtained from R & D Systems werereported by the supplier as >97% pure on SDS-PAGE (sodium dodecylsulphate-polyacrylamide gel electrophoresis) and biologically active ona bioassay specific for each ligand. The lyophilised chemokines weredissolved as a 100 μg/ml solution in a sterile phosphate-buffered saline(PBS) and this stock solution was stored at −20° C. in aliquots.Chemokines were diluted to the working concentration immediately beforeuse. All cell lines used in the present study were obtained from theATCC (Rockville, Md., USA).

Cloning and Sequencing

The mouse MOP020 clone was obtained by low stringency polymerase chainreaction, as described previously [24, 34], using genomic DNA astemplate. A human genomic DNA library (Stratagene, La Jolla, Calif.)constructed in the lambda DASH vector was screened at low stringency[39] with the MOP020 (511 bp) probe. The positive clones were purifiedto homogeneity and analysed by Southern blotting. The restriction map ofthe locus was determined and a relevant XbaI fragment of 4,400 bp wassubcloned in pBluescript SK+ (Stratagene). Sequencing was performed onboth strands after subcloning in M13mp derivatives, using fluorescentprimers and an automated DNA sequencer (Applied Biosystem 370A).Sequence handling and data analysis was carried out using theDNASIS/PROSIS software (Hitachi), and the GCG software package (GeneticsComputer Group, Wisconsin).

Expression in Cell Lines

The entire coding region was amplified by PCR as a 1056 bp fragment,using primers including respectively the BamBI and XbaI recognitionsequences, and cloned after restriction in the corresponding sites ofthe eukaryotic expression vector pcdna3 (Invitrogen, San Diego, Calif.).The resulting construct was verified by sequencing, and transfected inCHO-K1 cells as described [35]. Two days after transfection, selectionfor stably transfected cell lines was initiated by the addition of 400μg/ml G418 (Gibco), and resistant clones were isolated at day 10. CHO-K1cells were cultured using Ham's F12 medium, as previously described [35,11]. The expression of the active CCR5 receptor in the various cellclones was evaluated by measuring the specific transcript level byNorthern blotting, on total RNA prepared from the cells (see below).

Binding Assays

Stably transfected CHO-K1 cells expressing the active CCR5 receptor weregrown to confluence and detached from culture dishes by incubation inphosphate-buffered saline (PBS) supplemented with 1 Mm EDTA. Cells werecollected by low speed centrifugation and counted in a Neubaeur cell.Binding assays were performed in polyethylene minisorp tubes (Nunc) in afinal volume of 200 μl PBS containing 0.2% bovine serum albumin (BSA)and 10⁶ cells, in presence of [¹²⁵I]MIP-1α. Non specific binding wasdetermined by addition of 10 Nm unlabelled MIP-1α. The concentration oflabelled ligand was 0.4 Nm (around 100 000 cpm per tube). The incubationwas carried out for 2 hours at 4° C., and was stopped by the rapidaddition of 4 ml ice-cold buffer, and immediate collection of cells byvacuum filtration through GF/B glass fiber filters (Whatmann) pre-soakedin 0.5% polyethyleneinmine (Sigma). Filters were washed three times with4 ml ice-cold buffer and counted in a gamma counter.

Biological Activity

The CHO-K1 cell lines stably transfected with the pcdna3/CCR5 constructor wild type CHO-K1 cells (used as controls) were plated onto themembrane of Transwell cell capsules (Molecular Devices), at a density of2.5 10⁵ cells/well in Ham's F12 medium. The next day, the capsules weretransferred in a microphysiometer (Cytosensor, Molecular Devices), andthe cells were allowed to equilibrate for approximately two hours byperfusion of 1 Mm phosphate-buffered (Ph 7.4) RPMI-1640 mediumcontaining 0.2% BSA. Cells were then exposed to various chemokinesdiluted in the same medium, for a 2 min duration. Acidification rateswere measured at one minute intervals.

Northern Blotting

Total RNA was isolated from transfected CHO-K1 cell lines, from a panelof human cell lines of haematopoietic origin and from a panel of dogtissues, using the RNeasy kit (Qiagen). RNA samples (10 μg per lane)were denatured in presence of glyoxal [26], fractionated on a 1% agarosegel in a 10 Mm phosphate buffer (Ph 7.0), and transferred to nylonmembranes (Pall Biodyne A, Glen Cove, N.Y.) as described [42]. Afterbaking, the blots were prehybridized for 4 h at 42° C. in a solutionconsisting of 50% formamide, 5×Denhardt solution (1×Denhardt: 0.02%Ficoll, 0.02% polyvinylpyrolidone, 0.02% BSA), 5×SSPE (1×SSPE: 0.18 MNaCl, 10 Mm Na phosphate, 1 Mm EDTA Ph 8.3), 0.3% Sodium DodecylSulphate (SDS), 250 μg per ml denatured DNA from herring testes. DNAprobes were (α³²P)-labelled by random priming [14]. Hybridizations werecarried out for 12 h at 42° C. in the same solution containing 10%(wt/vol) dextran sulphate and the heat denatured probe. Filters werewashed up to 0.1×SSC (1×SSC: 150 Mm NaCl, 15 Mm Na Citrate Ph 7.0), 0.1%SDS at 60° C. and autoradiographed at −70° C. using Amersham β-maxfilms.

2. Results and Discussion

Cloning and Structural Analysis

The sequence homology characterising genes encoding G protein-coupledreceptors has allowed the cloning by low stringency polymerase chainreaction (PCR) of new members of this gene family [24, 34]. One of theclones amplified from mouse genomic DNA, named MOP020 presented strongsimilarities with characterised chemokine receptors, sharing 80%identity with the MCP-1 receptor (CCR2) [8], 65% identity with theMIP-1α/RANTES receptor (CCR1) [31], and 51% identity with IL-8 receptors[20, 30]. The clone was used as a probe to screen a human genomiclibrary. A total of 16 lambda phage clones were isolated. It wasinferred from the restriction pattern of each clone and from partialsequence data that all clones were belonging to a single contig in whichtwo different coding sequences were included. One of the codingsequences was identical to the reported cDNA encoding the CCR2 receptor[8, 44]. A 4.400 pb XbaI fragment of a representative clone containingthe second region of hybridization was subcloned in Pbluescript SK+.Sequencing revealed a novel gene, tentatively named CCR5, sharing 84%identity with the MOP020 probe, suggesting that MOP020 is the mouseortholog of CCR5. MOP020 does not correspond to any of the three mousechemoline receptor genes cloned recently [16], demonstrating theexistence of a fourth murine chemokine receptor.

The sequence of CCR5 revealed a single open reading frame of 352 codonsencoding a protein of 40,600 Da. The sequence surrounding the proposedinitiation codon is in agreement with the consensus as described byKozak [22], since the nucleotide in −3 is a purine. The hydropathyprofile of the deduced amino acid sequence is consistent with theexistence of 7 transmembrane segments. Alignment of the CCR5 amino acidsequence (SEQ ID NO:6) with that of other functionally characterisedhuman CC-chemokine receptors is represented in FIG. 2. The highestsimilarity is found with the CCR2 receptor (SEQ ID NO:8) [8] that shares75.8% identical residues. There is also 56.3% identity with the CCR1receptor (SEQ ID NO:7) [31], 58.4% with the CCR3(SEQ ID NO:9) [10], and49.1% with the CCR4 (SEQ ID NO:10) [37]. CCR5 represents therefore a newmember of the CC-chemokine receptor group [30]. Like the related CCR1and IL-8 receptors [20, 29, 31, 16] the coding region of CCR5 appears asintronless. From our partial sequencing data, the CCR2 gene is alsodevoid of intron in the first two thirds of its coding sequence.

Sequence similarities within the chemokine receptor family are higher inthe transmembrane-spanning domains, and in intracellular loops. As anexample, the identity score between CCR5 and CCR2 goes up to 92% whenconsidering the transmembrane segments only. Lower similarities arefound in the N-terminal extracellular domain, and in the extracellularloops. The N-terminal domain of the IL-8 and CCR2 receptors has beenshown to be essential for interaction with the ligand [19, 18]. Thevariability of this region among CC-chemokine receptors presumablycontributes to the specificity towards the various ligands of thefamily.

A single potential site for N-linked glycogylation wag identified in thethird extracellular loop of CCR5 (FIG. 1). No glycosylation site wasfound in the N-terminal domain of the receptor, where most Gprotein-coupled receptors are glycosylated. The other chemokinereceptors CCR1 and CCR2 present such an N-linked glycosylation site intheir N-terminal domain [31, 8]. By contrast, the CCR3 receptor [10]does not display glycosylation sites neither in the N-terminus, nor inextracellular loops. The active CCR5 receptor has four cysteines in itsextracellular segments, and all four are conserved in the other CC- andCXC-chemokine receptors (FIG. 2). The cysteines located in the first andsecond extracellular loops are present in most G protein-coupledreceptors, and are believed to form a disulphide bridge stabilising thereceptor structure [41]. The two other cysteines, in the N-terminalsegment, and in the third extracellular loop could similarly form astabilising bridge specific to the chemokine receptor family. Theintracellular domains of CCR5 do not include potential sites forphosphorylation by protein kinase C (PKC) or protein kinase A. PKCsites, involved in heterologous desensitisation are frequent in thethird intracellular loop and C-terminus of G protein-coupled receptors,CCR1 is also devoid of PKC sites. In contrast, all CC-chemokinereceptors, are rich in serine and threonine residues in the C-terminaldomain. These residues represent potential phosphorylation sites by thefamily of G protein-coupled receptor kinases, and are probably involvedin homologous desensitisation [41]. Five of these S/T residues areperfectly aligned in all five receptors (FIG. 2).

Physical Linkage of the CCR5 and CCR2 Genes

As stated above, the 16 clones isolated with the MOP020 probecorresponded to a single contig containing the CCR5 and CCR2 genes. Theorganisation of this contig was investigated in order to characterisethe physical linkage of the two receptor genes in the human genome. Acombination of restriction mapping, Southern blotting, fragmentsubcloning and partial sequencing allowed to determine the respectiveborders and overlaps of all clones. Out of the 16 clones, 9 turned outto be characterised by a specific restriction map, and theirorganisation is depicted in FIG. 3. Four of these clones (#11, 18, 21,22) contained the CCR2 gene alone, four clones (# 7, 13, 15, 16)contained the ChemR13 gene alone and one clone (#9) contains part ofboth coding sequences. The CCR2 and CCR5 genes are organised in tandem,CCR5 being located downstream of CCR2. The distance separating CCR2 andCCR5 open reading frames is 17.5 kb. The chromosomal localisation of thetandem is presently unknown. Other chemokine receptors have however beenlocated in the human genome: the CCR1 gene was localised by fluorescencein situ hybridization to the p21 region of human chromosome 3 [16]. Thetwo IL-8 receptor genes, and their pseudogene have been shown to beclustered on the human 2q34-q35 region [1].

Functional Expression and Pharmacology of the Active CCR5 Receptor

Stable CHO-K1 cell lines expressing the active CCR5 receptor wereestablished and were screened on the basis of the level of CCR5transcripts as determined by Northern blotting. Three clones wereselected and tested for biological responses in a microphysiometer,using various CC- and CXC-chemokines as potential agonists. Wild typeCHO-K1 cells were used as control to ensure that the observed responseswere specific for the transfected receptor, and did not result from theactivation of endogenous receptors. The microphysiometer allows the realtime detection of receptor activation, by measuring the modifications ofcell metabolism resulting from the stimulation of intracellular cascades[33]. Several studies have already demonstrated the potential ofmicrophysiometry in the field of chemokine receptors. Modifications ofmetabolic activity in human monocytes, in response CC-chemokines, weremonitored using this system [43]. Similarly, changes in theacidification rate of THP-1 cells (a human monocytic cell line) inresponse to MCP-1 and MCP-3 have been measured [36]. The estimation ofthe EC₅₀ for both proteins, using this procedure, was in agreement withthe values obtained by monitoring the intracellular calcium in otherstudies [8, 15].

Ligands belonging to the CC- and CXC-chemokine classes were tested onthe CCR5 transfected CHO-K1 cells. Whereas MIP-1α, MIP-1β and RANTESwere found to be potent activators of the new receptor (FIG. 4), theCC-chemokines MCP-1, MCP-2 and MCP-3, and the CXC-chemokines GROα andIL-8 had no effect on the metabolic activity, even at the highestconcentrations tested (30 Nm). The biological activity of one of thechemokines inducing no response on CCR5 (IL-8) could be demonstrated ona CHO-K1 cell line transfected with the IL-8A interleukin receptor(Mollereau et al., 1993): IL-8 produced a 160% increase in metabolicactivity as determined using the microphysiometer. The biologicalactivity of the MCP-2 and MCP-3 preparations as provided by J. Van Dammehave been widely documented [2, 40]. MIP-1α, MIP-1β and RANTES weretested on the wild type CHO-K1 cells, at a 30 Nm-concentration, and noneof them induced a metabolic response. On the CCR5 transfected CHO-K1cell line, all three active ligands (MIP-1α, MIP-1β and RANTES) caused arapid increase in acidification rate, reaching a maximum by the secondor third minute after perfusion of the ligand. The acidification ratereturned to basal level within 10 minutes. The timing of the cellularresponse is similar to that observed for chemokines on their naturalreceptors in human monocytes [43]. When agonists were applied repeatedlyto the same cells, the response was strongly reduced as compared to thefirst stimulation, suggesting the desensitisation of the receptor. Allmeasurements were therefore obtained on the first stimulation of eachcapsule.

The concentration-effect relation was evaluated for the three activeligands in the 0.3 to 30 Nm range (FIGS. 3B and C). The rank order ofpotency was MIP-1α>MIP-1β=RANTES. At 30 Nm concentrations, the effect ofMIP-1α appeared to saturate (at 156% of baseline level) while MIP-1β andRANTES were still in the ascending phase. Higher concentrations ofchemokines could however not be used. The EC50 was estimated around 3 Nmfor MIP-1α. The concentrations necessary for obtaining a biologicalresponse as determined by using the microphysiometer are in the samerange as those measured by intracellular calcium mobilisation for theCCR1 [31], the CCR2A and B [8], and the CCR3 [10] receptors. The ligandspecificity of CCR5 is similar to that reported for CCR3 [10]. CCR3 wasdescribed as the first cloned receptor responding to MIP-1β. However,MIP-1β at 10 Nm elicits a significant effect on the CCR5, while the sameconcentration is without effect on the CCR3 transfected cells [10].These data suggest that CCR5 could be a physiological receptor forMIP-1β.

Binding experiments using [¹²⁵I]-human MIP-1α as ligand did not allow todemonstrate specific binding to CCR53 expressing CHO-K1 cells, using asmuch as 0.4 Nm radioligand and 1 million transfected cells per tube.Failure to obtain binding data could be attributed to a relatively lowaffinity of the receptor for MIP-1α.

Northern Blotting Analysis

Northern blotting performed on a panel of dog tissues did not allow todetect transcripts for CCR5. Given the role of the chemokine receptorfamily in mediating chemoattraction and activation of various classes ofcells involved in inflammatory and immune responses, the probe was alsoused to detect specific transcripts in a panel of human cell lines ofhaematopoietic origin (FIG. 5). The panel included lymphoblastic (Raji)and T lymphoblastic (Jurkat) cell lines, promyeloblastic (KG-1A) andpromyelocytic (HL-60) cell lines, a monocytic (THP-1) cell line, anerythroleukemia (HEL 92.1.7) cell line, a megakaryoblastic (MEG-01) cellline, and a myelogenous leukaemia (K-562) cell line. Human peripheralblood mononuclear cells (PBMC), including mature monocytes andlymphocytes, were also tested. CCR5 transcripts (4.4 kb) could bedetected only in the KG-1A promyeloblastic cell line, but were not foundin the promyelocytic cell line HL-60, in PBMC, or in any of the othercell lines tested. These results suggest that the active CCR5 receptorcould be expressed in precursors of the granulocytic lineage.CC-chemokines have been reported to stimulate mature granulocytes [27,38, 23, 21]. However, recent data have also demonstrated a role of CC-and CXC-chemokines in the regulation of mouse and human myeloidprogenitor cell proliferation [6, 7].

CCR5 was shown to respond to MIP-1α, MIP-1β and RANTES, the threechemokines identified as the major HIV-suppressive factors produced byCD8⁺ T cells [9], and released in higher amounts by CD4⁺ T lymphocytesfrom uninfected but multiply exposed individuals [51]. CCR5 represents amajor co-receptor for macrophage-tropic (M-tropic) HIV-1 primaryisolates and strains [45, 50]. M-tropic strains predominate during theasymptomatic phase of the disease in infected individuals, and areconsidered as responsible for HIV-1 transmission. Strains adapted forgrowth in tranformed T-cell lines (T-tropic strains) use as aco-receptor LESTR (or fusin) [50], an orphan receptor also belonging tothe chemokine receptor family, but not yet characterised functionally[21, 52, 53]. Dual-tropic viruses, which may represent transitionalforms of the virus in late stages of infection [54] are shown to useboth CCR5 and LESTR as co-receptors, as well as the CC-chemolinereceptors CCR2b and CCR3 [47]. The broad spectrum of co-receptor usageof dual-tropic viruses suggests that within infected individuals, thevirus may evolve at least in part from selection by a variety ofco-receptors expressed on different cell types.

Identification of an Inactive ACCR5 Receptor

It is known that some individuals remain uninfected despite repeatedexposure to HIV-1 [55, 56, 51]. A proportion of these exposed-uninfectedindividuals results from the relatively low risk of contamination aftera single contact with the virus, but it has been postulated that trulyresistant individuals do exist. In fact, CD4⁺ lymphocytes isolated fromexposed-uninfected individualg are highly resistant to infection byprimary M-tropic, but not T-tropic HIV-1 strains. Also, peripheral bloodmononuclear cells (PBMC) from different donors are not infected equallywith various HIV-1 strains [57-59]. Given the key role played by CCR5 inthe fusion event that mediates infection by M-tropic viruses, it ispostulated that variants of CCR5 could be responsible for the relativeor absolute resistance to HIV-1 infection exhibited by some individuals,and possibly for the variability of disease progression in infectedpatients [66]. The Inventors selected three HIV-1 infected patientsknown to be slow progressors, and four seronegative individuals ascontrols; the fall coding region of their CCR5 gene was amplified by PCRand sequenced. Unexpectedly, one of the slow progressors, but also twoof the uninfected controls, exhibited heterozygosity at the CCR5 locusfor a biallelic polymorphism. The frequent allele corresponded to thepublished CCR5 sequence, while the minor one displayed a 32 bp deletionwithin the coding sequence, in a region corresponding to the secondextracellular loop of the receptor (FIG. 6). The FIG. 6 is the structureof the mutant form of human CC-chemokine receptor 5. a, The amino acidsequence of the non-functional Δccr5 protein (SEQ ID NO:6) isrepresented. The transmembrane organisation is given by analogy with thepredicted transmembrane structure of the wild-type CCR5. Amino acidsrepresented in black correspond to unnatural residues resulting from theframe shift caused by the deletion. The mutant protein lacks the lastthree transmembrane segments of CCR5, as well as the regions involved inG protein-coupling. b, Nucleotide sequence of the CCR5 gene surroundingthe deleted region (SEQ ID NO:12), and translation into the normalreceptor (top) (SEQ ID NO:11) or the truncated mutant (Δccr5, bottom)(SEQ ID NO:13). The 10-bp direct repeat is represented in italics. Thefull size coding region of the CCR5 gene was amplified by PCR, using5′-TCGAGGATCCAAGATGGATTATCAAGT-3′ (SEQ ID NO:14) and5′-CTGATCTAGAGCCATGTGCACAACTCT-3′ (SEQ ID NO:15) as forward and reverseprimers respectively. The PCR products were sequenced on both strandsusing the same oligonucleotides as primers, as well as internal primers,and fluorochrome-labelled dideoxynucleotides as terminators. Thesequencing products were run on an Applied Biosystem sequencer, andambiguous positions were searched along the coding sequence. When thepresence of a deletion was suspected from direct sequencing, the PCRproducts were cloned after restriction with BamHI and XbaI endonucleasesinto pcdna3. Several clones were sequenced to confirm the deletion. Thedeletion was identical in three unrelated individuals investigated bysequencing.

Cloning of the PCR product and sequencing of several clones confirmedthe deletion. The deletion causes a frame shift, which is expected toresult in premature termination of translation. The protein encoded bythis mutant allele (Δccr5) therefore lacks the last three transmembranesegments of the receptor. A 10-bp direct repeat flanking the deletedregion (FIG. 6b) on both sides is expected to have promoted therecombination event leading to the deletion. Numerous mutagenesisstudies performed on various classes of G protein-coupled receptors,including chemokine receptors, makes it clear that such a truncatedprotein is certainly not functional in terms of chemokine-induced signaltransduction: it lacks the third intracellular loop and C-terminalcytoplasmic domains, the two regions involved primarily in G proteincoupling [41]. In order to test whether the truncated protein was ableto function as a HIV-1 co-receptor, the Inventors tested its ability tosupport membrane fusion by both primary M-tropic and dual-tropic virusENV proteins. The recombinant protein was expressed in quail QT6 cellstogether with human CD4. The QT6 cells were then mixed with HeLa cellsexpressing the indicated viral ENV protein and the extent of cell-cellfusion measured using a sensitive and quantitative gene-reporter assay.In contrast to wild-type CCR5, the truncated receptor did not allowfusion with cells expressing the ENV protein from either M-tropic ordual-tropic viruses (FIG. 7). The FIG. 7 represents the quantificationof ENV protein-mediated fusion by luciferase assay. To quantifycell-cell fusion events, Japanese quail QT6 fibrosarcoma cells weretransfected or cotransfected as indicated with the pcdna3 vector(Invitrogen) containing the coding sequence for wild-type CCR5, thetruncated cers mutant, the CCR2b or the Duffy chemokine receptors, orwith the PCDNA3 vector alone. The target cells were also transfectedwith human CD4 expressed from the CMV promoter and the luciferase geneunder the control of the T7 promoter. HeLa effector cells were infected(MOI=10) with vaccinia vectors expressing T7-polymerase (vTF1.1) andeither the JR-FL (vCB28) or 89.6 (vBD3) envelope proteins. Theluciferase activity resulting from cell fusion is expressed as thepercentage of the activity (in relative light units) obtained forwild-type CCR5. All transfections were performed with an identicalquantity of plasmid DNA using pcdna3 as carrier when necessary. Toinitiate fusion, target and effector cells were mixed in 24 well platesat 37° C. in the presence of ara-C and rifampicin, and allowed to fusefor 8 hours. Cells were lysed in 150 μl of reporter lysis bufferPromega) and assayed for luciferase activity according to themanufacturer's instructions (Promega).

Coexpression of Δccr5 with wild-type CCR5 consistently reduced theefficiency of fusion for both JR-FL and 89.6 envelopes, as compared withCCR5 alone. Whether this in vitro inhibitory effect (not shared by thechemokine receptor Duffy, used as control) also occurs in vivo ispresently not known. Coexpression with the CCR2b receptor [31], which isthe CC-chemokine receptor most closely related to CCR5 but does notpromote fusion by M-tropic HIV-1strains [48], did not rescue themutation by formation of a hybrid molecule (FIG. 7).

The FIG. 8 represents genotyping of individuals by PCR and segregationof the CCR5 alleles in CEPH families. a, Autoradiography illustratingthe pattern resulting from PCR amplification and EcoRI cleavage forindividuals homozygous for the wild-type CCR5 allele (CCR5/CCR5), thenull ΔΔccr5 allele (Δccr5/Δccr5), and for heterozygotes (CCR5/Δccr5). A735 bp PCR product is cleaved into a common band of 332 bp for bothalleles, and into 403 and 371 bp bands for the wild-type and mutantalleles, respectively. b, Segregation of the CCR5 alleles in twoinformative families of the CEPH. Half-black and white symbols representheterozygotes and wild-type homozygotes, respectively. For a fewindividuals in the pedigrees, DNA was not available (ND: notdetermined). PCRs were performed on genomic DNA samples, using5′-CCTGGCTGTCGTCCATGCTG-3′ (SEQ ID NO:16) and5′-CTGATCTAGAGCCATGTGCACAACTCT-3′ (SEQ ID NO:17) as forward and reverseprimers respectively. Reaction mixtures consisted in 30 μl of 10 MmTris-Hcl buffer Ph 8.0, containing 50 Mm Kcl, 0.75 Mm MgCl₂, 0.2 MmdCTP, dGTP and dTTP, 0.1 Mm dATP, 0.5 μi [α-³²P]-DATP, 0.01% gelatine,5% DMSO, 200 ng target DNA, 60 ng of each of the primers and 1.5 U Taqpolymerase. PCR conditions were: 93° C. for 2 min 30; 93° C. for 1 min,60° C. for 1 min, 72° C. for 1 min, 30 cycles; 72° C. for 6 min. Afterthe PCR reaction, the samples were incubated for 60 min at 37° C. with10 U EcoRI, and 2 μl of the denatured reaction mixture was applied ontoa denaturing 5% polyacrylamide gel containing 35% formamide and 5.6 Murea. Bands were detected by autoradiography.

Based on the 14 chromosomes tested in the first experiment, the deletedΔccr5 allele appeared rather frequent in the Caucasian population. Theaccurate frequency was further estimated by testing (FIG. 8a) a largecohort of Caucasian individuals, including unrelated members of the CEPH(Centre d'Etude des Polymorphismes Humains) families, part of the IRIBHNstaff, and a bank of anonymous DNA samples from healthy individualscollected by the Genetics Department of the Erasme Hospital in Brussels.From a total of more than 700 healthy individuals, the allelefrequencies were found to be 0.908 for the wild-type allele, and 0.092for the mutant allele (Table I). The genotype frequencies observed inthe population were not significantly different from the expectedHardy-Weinberg distribution (CCR5/CCR5: 0.827 vs 0.824; CCR5/Δccr5:0.162 vs 0.167; Δccr5/Δccr5: 0.011 vs 0.008, p>0.999), suggesting thatthe null allele has no drastic effect on fitness. Using two informativeCEPH families, it was confirmed that the wild-type CCR5 gene and itsΔccr5 variant were allelic, and segregated in a normal mendelian fashion(FIG. 8b). Interestingly, a cohort of 124 DNA samples originating fromCentral Africa (collected from Zaire, Burkina Fasso, Cameroun, Senegaland Benin) and Japan did not reveal a single Δccr5 mutant allele,suggesting that this allele is either absent or very rare in Asian,African black populations (Table I).

The consequences of the existence of a null allele of CCR5 in the normalCaucasian population were then considered in terms of susceptibility toinfection by HIV-1. If, as it is predicted, CCR5 plays a major (notredundant) role in the entry of most primary virus strains into cells,then Δccr5/Δccr5 individuals should be particularly resistant to HIV-1challenge, both in vitro and in vivo. The frequency of the Δccr5/Δccr5genotype should therefore be significantly lower in HIV-1 infectedpatients, and increased in exposed-uninfected individuals. Also, ifheterozygotes have a statistical advantage due to the lower number offunctional receptors on their white blood cells, or to the possibledominant-negative properties of the mutant allele, the frequency ofheterozygotes (and mutant alleles) should be decreased in HIV-infectedpopulations. These hypotheses were tested by genotyping a large numberof seropositive Caucasian individuals (n=645) belonging to cohortsoriginating from various hospitals from Brussels, Liege and Paris (TableI). Indeed, it was found that within this large series, the frequency ofthe null Δccr5 allele was significantly reduced from 0.092 to 0.053(p<10⁻⁵). The frequency of heterozygotes was also reduced from 0.162 to0.106 (p<0.001) and not a single Δccr5/Δccr5 individual could be found(p<0.01).

Altogether, functional and statistical data suggest that CCR5 is indeedthe major co-receptor responsible for natural infection by M-tropicHIV-1 strains. Individuals homozygous for the null Δccr5 allele (about1% of the Caucasian population) have apparently a strong resistance toinfection. It is unclear at this point whether resistance to HIV-1 isabsolute or relative, and whether resistance will vary depending on themode of viral contamination. Larger cohorts of seropositive individualswill have to be tested in order to clarify this point. Heterozygoteshave a milder though significant advantage: assuming an equalprobability of contact with HIV, it can be inferred from Table I thatheterozygotes have a 39% reduction in their likeliness of becomingseropositive, as compared to individuals homozygous for the wild-typeCCR5 allele. Both a decrease in functional CCR5 receptor number, and adominant-negative effect of Δccr5 in vivo, comparable to what isobserved in the in vitro experiments (FIG. 7) are possible explanationsfor this relative protection. The mutant allele, which can be regardedas a natural knock-out in human, is not accompanied by an obviousphenotype in homozygous individuals. Nevertheless, the lack of overtphenotype, taken together with the relative protection thatcharacterises heterozygous subjects, suggests that pharmacologicalagents that selectively block the ability of HIV-1 to utilise CCR5 as acofactor, could be effective in preventing HIV-1 infection, and would bepredicted not be associated with major side effects resulting from CCR5inactivation. These pharmaceutical agents could be used with othercompounds which are able to block other chemokine receptors used asco-receptors by some HIV-primary isolates in order to infect other cells[47]. The prevalence of the null allele in the Caucasian populationraises the question of whether pandemia of HIV (or related viruses usingthe same co-receptor) have contributed during mankind's evolution tostabilige by selection the mutant ccr5 allele at such a high frequency.

Production of Antibodies Anti-CCR5

Antibodies were produced by genetic immunisation. Six week old femalesbalb/c mice were used. DNA coding for the human CCR5 receptor wasinserted in the expression vector pcdna3 under the control of the CMVpromotor and 100 μg DNA was injected in the anterior tibial muscle, fivedays after pre-treatment of this muscle with cardiotoxine (from venom ofNaja Nigricolis). Injections were repeated twice at three weekintervals. Fifteen days after the last injection, blood was taken fromeach animal and sera were tested for the presence of anti-CCR5antibodies.

Test of Sera using Fluorescence Activated Cell Sorter (FACS)

Sera were tested by fluorescence activated cell sorting usingrecombinant CHO cells expressing the CCR5 receptor. Briefly, cells weredetached using a PBS-EDTA-EGTA solution and incubated into PBS-BSAmedium for 30 minutes at room temperature with 5 μl serum on the basisof 100,000 cells per tube. Cells were then washed and incubated for 30minutes in ice together with anti-mouse antibody labelled withfluorescein. Cells were washed, taken up into 200 μl of a PBS-BSAsolution and fluorescence was analysed by FACS (FACSCAN,Becton-Dickinson). 10,000 cells were counted. Wild type CHO orrecombinant CHO cells expressing the human CCR2b receptor were used ascontrols.

When tested by FACS analysis 2 weeks after the last injection (FIG. 9),all the sera from mice immunised with CCR5 cDNA, clearly recognised thenative receptor expressed on CHO cells (mean of fluorescence=200),without significant cross reaction with control cells expressing CCR2b(mean of fluorescence=20).

Sera were tested on either a CHO cell line expressing high level of CCR5receptor (black histogram) or a CHO cell line expressing CCR2b receptor(white histogram) as negative control. Each serum was testedindividually.

Antibodies Anti-CCR5 and HIV Infectivity

Peripheral blood mononuclear cells (PBMC) from one donor homozygous fromwild type CCR5 gene, were isolated and cultivated 3 days in presence ofPHA.

On day 4, 800 μl of cells (10⁵ cells/ml) were incubated with 8 μl ofsera from mice immunised with CCR5 cDNA, 30 minutes at 37° C. 1 ml ofviral solution (JRCSF HIV strain) is then added and incubated during 2hours. Cells were then washed twice and cultivated during 15 days.

Aliquot of medium is taken at days 0, 4, 7, 10 and 14 and the dosage ofantigen p24 is performed.

14 days after the beginning of the experiment, one serum (serum B0)totally block the production of p24, indicating its ability to block theinfection of the lymphocytes by this HIV strain (FIG. 10). Other serumsalso exhibit a partial or total effect on this infection (serum A2 andB1). All the other sera did not show any effect on this infection.

Production of Monoclonal Antibodies

Mice with the highest title of CCR5 antibodies were selected formonoclonal antibodies production and injected intravenously with 10⁷recombinant CHO-K1 cells expressing human CCR5 receptors. Three dayslater, animals were sacrificed and fusion of splenic cells or cells fromlymph nodes near the site of injection with SP2/0 myeloma cells, wereperformed. Fusion protocol used was that of Galfre et al. (Nature 266,550 (1977)). A selective HAT (hypoxanthine/aminopterin/thymidin) mediumis used to select hybridomas and their supernatants are tested by FACSusing recombinant CHO cells expressing the human CCR5 receptor, as itwas done for the sera. Positives hybridomas are then cloned by limiteddilution. Clones that are shown positive by FACS analyses are thenexpanded and produced in ascites in balb/C mice. TABLE (KOA-3834.2)

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62. Nussbaum et al. (1994) J. Virol. 68, 5411-5422.

17 792 base pairs nucleic acid single linear DNA (genomic) CDS 240..7911 GAATTCCCCC AACAGAGCCA AGCTCTCCAT CTAGTGGACA GGGAAGCTAG CAGCAAACCT 60TCCCTTCACT ACAAAACTTC ATTGCTTGGC CAAAAAGAGA GTTAATTCAA TGTAGACATC 120TATGTAGGCA ATTAAAAACC TATTGATGTA TAAAACAGTT TGCATTCATG GAGGGCAACT 180AAATACATTC TAGGACTTTA TAAAAGATCA CTTTTTATTT ATGCACAGGG TGGAACAAG 239 ATGGAT TAT CAA GTG TCA AGT CCA ATC TAT GAC ATC AAT TAT TAT ACA 287 Met AspTyr Gln Val Ser Ser Pro Ile Tyr Asp Ile Asn Tyr Tyr Thr 1 5 10 15 TCGGAG CCC TGC CAA AAA ATC AAT GTG AAG CAA ATC GCA GCC CGC CTC 335 Ser GluPro Cys Gln Lys Ile Asn Val Lys Gln Ile Ala Ala Arg Leu 20 25 30 CTG CCTCCG CTC TAC TCA CTG GTG TTC ATC TTT GGT TTT GTG GGC AAC 383 Leu Pro ProLeu Tyr Ser Leu Val Phe Ile Phe Gly Phe Val Gly Asn 35 40 45 ATG CTG GTCATC CTC ATC CTG ATA AAC TGC AAA AGG CTG AAG AGC ATG 431 Met Leu Val IleLeu Ile Leu Ile Asn Cys Lys Arg Leu Lys Ser Met 50 55 60 ACT GAC ATC TACCTG CTC AAC CTG GCC ATC TCT GAC CTG TTT TTC CTT 479 Thr Asp Ile Tyr LeuLeu Asn Leu Ala Ile Ser Asp Leu Phe Phe Leu 65 70 75 80 CTT ACT GTC CCCTTC TGG GCT CAC TAT GCT GCC GCC CAG TGG GAC TTT 527 Leu Thr Val Pro PheTrp Ala His Tyr Ala Ala Ala Gln Trp Asp Phe 85 90 95 GGA AAT ACA ATG TGTCAA CTC TTG ACA GGG CTC TAT TTT ATA GGC TTC 575 Gly Asn Thr Met Cys GlnLeu Leu Thr Gly Leu Tyr Phe Ile Gly Phe 100 105 110 TTC TCT GGA ATC TTCTTC ATC ATC CTC CTG ACA ATC GAT AGG TAC CTG 623 Phe Ser Gly Ile Phe PheIle Ile Leu Leu Thr Ile Asp Arg Tyr Leu 115 120 125 GCT GTC GTC CAT GCTGTG TTT GCT TTA AAA GCC AGG ACG GTC ACC TTT 671 Ala Val Val His Ala ValPhe Ala Leu Lys Ala Arg Thr Val Thr Phe 130 135 140 GGG GTG GTG ACA AGTGTG ATC ACT TGG GTG GTG GCT GTG TTT GCG TCT 719 Gly Val Val Thr Ser ValIle Thr Trp Val Val Ala Val Phe Ala Ser 145 150 155 160 CTC CCA GGA ATCATC TTT ACC AGA TCT CAA AAA GAA GGT CTT CAT TAC 767 Leu Pro Gly Ile IlePhe Thr Arg Ser Gln Lys Glu Gly Leu His Tyr 165 170 175 ACC TGC AGC TCTCAT TTT CCA TAC A 792 Thr Cys Ser Ser His Phe Pro Tyr 180 1477 basepairs nucleic acid single linear DNA (genomic) CDS 240..1295 2GAATTCCCCC AACAGAGCCA AGCTCTCCAT CTAGTGGACA GGGAAGCTAG CAGCAAACCT 60TCCCTTCACT ACAAAACTTC ATTGCTTGGC CAAAAAGAGA GTTAATTCAA TGTAGACATC 120TATGTAGGCA ATTAAAAACC TATTGATGTA TAAAACAGTT TGCATTCATG GAGGGCAACT 180AAATACATTC TAGGACTTTA TAAAAGATCA CTTTTTATTT ATGCACAGGG TGGAACAAG 239 ATGGAT TAT CAA GTG TCA AGT CCA ATC TAT GAC ATC AAT TAT TAT ACA 287 Met AspTyr Gln Val Ser Ser Pro Ile Tyr Asp Ile Asn Tyr Tyr Thr 1 5 10 15 TCGGAG CCC TGC CAA AAA ATC AAT GTG AAG CAA ATC GCA GCC CGC CTC 335 Ser GluPro Cys Gln Lys Ile Asn Val Lys Gln Ile Ala Ala Arg Leu 20 25 30 CTG CCTCCG CTC TAC TCA CTG GTG TTC ATC TTT GGT TTT GTG GGC AAC 383 Leu Pro ProLeu Tyr Ser Leu Val Phe Ile Phe Gly Phe Val Gly Asn 35 40 45 ATG CTG GTCATC CTC ATC CTG ATA AAC TGC AAA AGG CTG AAG AGC ATG 431 Met Leu Val IleLeu Ile Leu Ile Asn Cys Lys Arg Leu Lys Ser Met 50 55 60 ACT GAC ATC TACCTG CTC AAC CTG GCC ATC TCT GAC CTG TTT TTC CTT 479 Thr Asp Ile Tyr LeuLeu Asn Leu Ala Ile Ser Asp Leu Phe Phe Leu 65 70 75 80 CTT ACT GTC CCCTTC TGG GCT CAC TAT GCT GCC GCC CAG TGG GAC TTT 527 Leu Thr Val Pro PheTrp Ala His Tyr Ala Ala Ala Gln Trp Asp Phe 85 90 95 GGA AAT ACA ATG TGTCAA CTC TTG ACA GGG CTC TAT TTT ATA GGC TTC 575 Gly Asn Thr Met Cys GlnLeu Leu Thr Gly Leu Tyr Phe Ile Gly Phe 100 105 110 TTC TCT GGA ATC TTCTTC ATC ATC CTC CTG ACA ATC GAT AGG TAC CTG 623 Phe Ser Gly Ile Phe PheIle Ile Leu Leu Thr Ile Asp Arg Tyr Leu 115 120 125 GCT GTC GTC CAT GCTGTG TTT GCT TTA AAA GCC AGG ACG GTC ACC TTT 671 Ala Val Val His Ala ValPhe Ala Leu Lys Ala Arg Thr Val Thr Phe 130 135 140 GGG GTG GTG ACA AGTGTG ATC ACT TGG GTG GTG GCT GTG TTT GCG TCT 719 Gly Val Val Thr Ser ValIle Thr Trp Val Val Ala Val Phe Ala Ser 145 150 155 160 CTC CCA GGA ATCATC TTT ACC AGA TCT CAA AAA GAA GGT CTT CAT TAC 767 Leu Pro Gly Ile IlePhe Thr Arg Ser Gln Lys Glu Gly Leu His Tyr 165 170 175 ACC TGC AGC TCTCAT TTT CCA TAC AGT CAG TAT CAA TTC TGG AAG AAT 815 Thr Cys Ser Ser HisPhe Pro Tyr Ser Gln Tyr Gln Phe Trp Lys Asn 180 185 190 TTC CAG ACA TTAAAG ATA GTC ATC TTG GGG CTG GTC CTG CCG CTG CTT 863 Phe Gln Thr Leu LysIle Val Ile Leu Gly Leu Val Leu Pro Leu Leu 195 200 205 GTC ATG GTC ATCTGC TAC TCG GGA ATC CTA AAA ACT CTG CTT CGG TGT 911 Val Met Val Ile CysTyr Ser Gly Ile Leu Lys Thr Leu Leu Arg Cys 210 215 220 CGA AAT GAG AAGAAG AGG CAC AGG GCT GTG AGG CTT ATC TTC ACC ATC 959 Arg Asn Glu Lys LysArg His Arg Ala Val Arg Leu Ile Phe Thr Ile 225 230 235 240 ATG ATT GTTTAT TTT CTC TTC TGG GCT CCC TAC AAC ATT GTC CTT CTC 1007 Met Ile Val TyrPhe Leu Phe Trp Ala Pro Tyr Asn Ile Val Leu Leu 245 250 255 CTG AAC ACCTTC CAG GAA TTC TTT GGC CTG AAT AAT TGC AGT AGC TCT 1055 Leu Asn Thr PheGln Glu Phe Phe Gly Leu Asn Asn Cys Ser Ser Ser 260 265 270 AAC AGG TTGGAC CAA GCT ATG CAG GTG ACA GAG ACT CTT GGG ATG ACG 1103 Asn Arg Leu AspGln Ala Met Gln Val Thr Glu Thr Leu Gly Met Thr 275 280 285 CAC TGC TGCATC AAC CCC ATC ATC TAT GCC TTT GTC GGG GAG AAG TTC 1151 His Cys Cys IleAsn Pro Ile Ile Tyr Ala Phe Val Gly Glu Lys Phe 290 295 300 AGA AAC TACCTC TTA GTC TTC TTC CAA AAG CAC ATT GCC AAA CGC TTC 1199 Arg Asn Tyr LeuLeu Val Phe Phe Gln Lys His Ile Ala Lys Arg Phe 305 310 315 320 TGC AAATGC TGT TCT ATT TTC CAG CAA GAG GCT CCC GAG CGA GCA AGC 1247 Cys Lys CysCys Ser Ile Phe Gln Gln Glu Ala Pro Glu Arg Ala Ser 325 330 335 TCA GTTTAC ACC CGA TCC ACT GGG GAG CAG GAA ATA TCT GTG GGC TTG 1295 Ser Val TyrThr Arg Ser Thr Gly Glu Gln Glu Ile Ser Val Gly Leu 340 345 350TGACACGGAC TCAAGTGGGC TGGTGACCCA GTCAGAGTTG TGCACATGGC TTAGTTTTCA 1355TACACAGCCT GGGCTGGGGG TNGGTTGGNN GAGGTCTTTT TTAAAAGGAA GTTACTGTTA 1415TAGAGGGTCT AAGATTCATC CATTTATTTG GCATCTGTTT AAAGTAGATT AGATCCGAAT 1475TC 1477 1442 base pairs nucleic acid single linear DNA (genomic) CDS240..884 3 GAATTCCCCC AACAGAGCCA AGCTCTCCAT CTAGTGGACA GGGAAGCTAGCAGCAAACCT 60 TCCCTTCACT ACAAAACTTC ATTGCTTGGC CAAAAAGAGA GTTAATTCAATGTAGACATC 120 TATGTAGGCA ATTAAAAACC TATTGATGTA TAAAACAGTT TGCATTCATGGAGGGCAACT 180 AAATACATTC TAGGACTTTA TAAAAGATCA CTTTTTATTT ATGCACAGGGTGGAACAAG 239 ATG GAT TAT CAA GTG TCA AGT CCA ATC TAT GAC ATC AAT TATTAT ACA 287 Met Asp Tyr Gln Val Ser Ser Pro Ile Tyr Asp Ile Asn Tyr TyrThr 1 5 10 15 TCG GAG CCC TGC CAA AAA ATC AAT GTG AAG CAA ATC GCA GCCCGC CTC 335 Ser Glu Pro Cys Gln Lys Ile Asn Val Lys Gln Ile Ala Ala ArgLeu 20 25 30 CTG CCT CCG CTC TAC TCA CTG GTG TTC ATC TTT GGT TTT GTG GGCAAC 383 Leu Pro Pro Leu Tyr Ser Leu Val Phe Ile Phe Gly Phe Val Gly Asn35 40 45 ATG CTG GTC ATC CTC ATC CTG ATA AAC TGC AAA AGG CTG AAG AGC ATG431 Met Leu Val Ile Leu Ile Leu Ile Asn Cys Lys Arg Leu Lys Ser Met 5055 60 ACT GAC ATC TAC CTG CTC AAC CTG GCC ATC TCT GAC CTG TTT TTC CTT479 Thr Asp Ile Tyr Leu Leu Asn Leu Ala Ile Ser Asp Leu Phe Phe Leu 6570 75 80 CTT ACT GTC CCC TTC TGG GCT CAC TAT GCT GCC GCC CAG TGG GAC TTT527 Leu Thr Val Pro Phe Trp Ala His Tyr Ala Ala Ala Gln Trp Asp Phe 8590 95 GGA AAT ACA ATG TGT CAA CTC TTG ACA GGG CTC TAT TTT ATA GGC TTC575 Gly Asn Thr Met Cys Gln Leu Leu Thr Gly Leu Tyr Phe Ile Gly Phe 100105 110 TTC TCT GGA ATC TTC TTC ATC ATC CTC CTG ACA ATC GAT AGG TAC CTG623 Phe Ser Gly Ile Phe Phe Ile Ile Leu Leu Thr Ile Asp Arg Tyr Leu 115120 125 GCT GTC GTC CAT GCT GTG TTT GCT TTA AAA GCC AGG ACG GTC ACC TTT671 Ala Val Val His Ala Val Phe Ala Leu Lys Ala Arg Thr Val Thr Phe 130135 140 GGG GTG GTG ACA AGT GTG ATC ACT TGG GTG GTG GCT GTG TTT GCG TCT719 Gly Val Val Thr Ser Val Ile Thr Trp Val Val Ala Val Phe Ala Ser 145150 155 160 CTC CCA GGA ATC ATC TTT ACC AGA TCT CAA AAA GAA GGT CTT CATTAC 767 Leu Pro Gly Ile Ile Phe Thr Arg Ser Gln Lys Glu Gly Leu His Tyr165 170 175 ACC TGC AGC TCT CAT TTT CCA TAC ATT AAA GAT AGT CAT CTT GGGGCT 815 Thr Cys Ser Ser His Phe Pro Tyr Ile Lys Asp Ser His Leu Gly Ala180 185 190 GGT CCT GCC GCT GCT TGT CAT GGT CAT CTG CTA CTC GGG AAT CCTAAA 863 Gly Pro Ala Ala Ala Cys His Gly His Leu Leu Leu Gly Asn Pro Lys195 200 205 AAC TCT GCT TCG GTG TCG AAA TGAGAAGAAG AGGCACAGGG CTGTGAGGCT914 Asn Ser Ala Ser Val Ser Lys 210 215 TATCTTCACC ATCATGATTG TTTATTTTCTCTTCTGGGCT CCCTACAACA TTGTCCTTCT 974 CCTGAACACC TTCCAGGAAT TCTTTGGCCTGAATAATTGC AGTAGCTCTA ACAGGTTGGA 1034 CCAAGCTATG CAGGTGACAG AGACTCTTGGGATGACGCAC TGCTGCATCA ACCCCATCAT 1094 CTATGCCTTT GTCGGGGAGA AGTTCAGAAACTACCTCTTA GTCTTCTTCC AAAAGCACAT 1154 TGCCAAACGC TTCTGCAAAT GCTGTTCTATTTTCCAGCAA GAGGCTCCCG AGCGAGCAAG 1214 CTCAGTTTAC ACCCGATCCA CTGGGGAGCAGGAAATATCT GTGGGCTTGT GACACGGACT 1274 CAAGTGGGCT GGTGACCCAG TCAGAGTTGTGCACATGGCT TAGTTTTCAT ACACAGCCTG 1334 GGCTGGGGGT GGTTGGGAGG TCTTTTTTAAAAGGAAGTTA CTGTTATAGA GGGTCTAAGA 1394 TTCATCCATT TATTTGGCAT CTGTTTAAAGTAGATTAGAT CCGAATTC 1442 184 amino acids amino acid linear protein 4 MetAsp Tyr Gln Val Ser Ser Pro Ile Tyr Asp Ile Asn Tyr Tyr Thr 1 5 10 15Ser Glu Pro Cys Gln Lys Ile Asn Val Lys Gln Ile Ala Ala Arg Leu 20 25 30Leu Pro Pro Leu Tyr Ser Leu Val Phe Ile Phe Gly Phe Val Gly Asn 35 40 45Met Leu Val Ile Leu Ile Leu Ile Asn Cys Lys Arg Leu Lys Ser Met 50 55 60Thr Asp Ile Tyr Leu Leu Asn Leu Ala Ile Ser Asp Leu Phe Phe Leu 65 70 7580 Leu Thr Val Pro Phe Trp Ala His Tyr Ala Ala Ala Gln Trp Asp Phe 85 9095 Gly Asn Thr Met Cys Gln Leu Leu Thr Gly Leu Tyr Phe Ile Gly Phe 100105 110 Phe Ser Gly Ile Phe Phe Ile Ile Leu Leu Thr Ile Asp Arg Tyr Leu115 120 125 Ala Val Val His Ala Val Phe Ala Leu Lys Ala Arg Thr Val ThrPhe 130 135 140 Gly Val Val Thr Ser Val Ile Thr Trp Val Val Ala Val PheAla Ser 145 150 155 160 Leu Pro Gly Ile Ile Phe Thr Arg Ser Gln Lys GluGly Leu His Tyr 165 170 175 Thr Cys Ser Ser His Phe Pro Tyr 180 352amino acids amino acid linear protein 5 Met Asp Tyr Gln Val Ser Ser ProIle Tyr Asp Ile Asn Tyr Tyr Thr 1 5 10 15 Ser Glu Pro Cys Gln Lys IleAsn Val Lys Gln Ile Ala Ala Arg Leu 20 25 30 Leu Pro Pro Leu Tyr Ser LeuVal Phe Ile Phe Gly Phe Val Gly Asn 35 40 45 Met Leu Val Ile Leu Ile LeuIle Asn Cys Lys Arg Leu Lys Ser Met 50 55 60 Thr Asp Ile Tyr Leu Leu AsnLeu Ala Ile Ser Asp Leu Phe Phe Leu 65 70 75 80 Leu Thr Val Pro Phe TrpAla His Tyr Ala Ala Ala Gln Trp Asp Phe 85 90 95 Gly Asn Thr Met Cys GlnLeu Leu Thr Gly Leu Tyr Phe Ile Gly Phe 100 105 110 Phe Ser Gly Ile PhePhe Ile Ile Leu Leu Thr Ile Asp Arg Tyr Leu 115 120 125 Ala Val Val HisAla Val Phe Ala Leu Lys Ala Arg Thr Val Thr Phe 130 135 140 Gly Val ValThr Ser Val Ile Thr Trp Val Val Ala Val Phe Ala Ser 145 150 155 160 LeuPro Gly Ile Ile Phe Thr Arg Ser Gln Lys Glu Gly Leu His Tyr 165 170 175Thr Cys Ser Ser His Phe Pro Tyr Ser Gln Tyr Gln Phe Trp Lys Asn 180 185190 Phe Gln Thr Leu Lys Ile Val Ile Leu Gly Leu Val Leu Pro Leu Leu 195200 205 Val Met Val Ile Cys Tyr Ser Gly Ile Leu Lys Thr Leu Leu Arg Cys210 215 220 Arg Asn Glu Lys Lys Arg His Arg Ala Val Arg Leu Ile Phe ThrIle 225 230 235 240 Met Ile Val Tyr Phe Leu Phe Trp Ala Pro Tyr Asn IleVal Leu Leu 245 250 255 Leu Asn Thr Phe Gln Glu Phe Phe Gly Leu Asn AsnCys Ser Ser Ser 260 265 270 Asn Arg Leu Asp Gln Ala Met Gln Val Thr GluThr Leu Gly Met Thr 275 280 285 His Cys Cys Ile Asn Pro Ile Ile Tyr AlaPhe Val Gly Glu Lys Phe 290 295 300 Arg Asn Tyr Leu Leu Val Phe Phe GlnLys His Ile Ala Lys Arg Phe 305 310 315 320 Cys Lys Cys Cys Ser Ile PheGln Gln Glu Ala Pro Glu Arg Ala Ser 325 330 335 Ser Val Tyr Thr Arg SerThr Gly Glu Gln Glu Ile Ser Val Gly Leu 340 345 350 215 amino acidsamino acid linear protein 6 Met Asp Tyr Gln Val Ser Ser Pro Ile Tyr AspIle Asn Tyr Tyr Thr 1 5 10 15 Ser Glu Pro Cys Gln Lys Ile Asn Val LysGln Ile Ala Ala Arg Leu 20 25 30 Leu Pro Pro Leu Tyr Ser Leu Val Phe IlePhe Gly Phe Val Gly Asn 35 40 45 Met Leu Val Ile Leu Ile Leu Ile Asn CysLys Arg Leu Lys Ser Met 50 55 60 Thr Asp Ile Tyr Leu Leu Asn Leu Ala IleSer Asp Leu Phe Phe Leu 65 70 75 80 Leu Thr Val Pro Phe Trp Ala His TyrAla Ala Ala Gln Trp Asp Phe 85 90 95 Gly Asn Thr Met Cys Gln Leu Leu ThrGly Leu Tyr Phe Ile Gly Phe 100 105 110 Phe Ser Gly Ile Phe Phe Ile IleLeu Leu Thr Ile Asp Arg Tyr Leu 115 120 125 Ala Val Val His Ala Val PheAla Leu Lys Ala Arg Thr Val Thr Phe 130 135 140 Gly Val Val Thr Ser ValIle Thr Trp Val Val Ala Val Phe Ala Ser 145 150 155 160 Leu Pro Gly IleIle Phe Thr Arg Ser Gln Lys Glu Gly Leu His Tyr 165 170 175 Thr Cys SerSer His Phe Pro Tyr Ile Lys Asp Ser His Leu Gly Ala 180 185 190 Gly ProAla Ala Ala Cys His Gly His Leu Leu Leu Gly Asn Pro Lys 195 200 205 AsnSer Ala Ser Val Ser Lys 210 215 360 amino acids amino acid single linearNone 7 Met Leu Ser Thr Ser Arg Ser Arg Phe Ile Arg Asn Thr Asn Glu Ser 15 10 15 Gly Glu Glu Val Thr Thr Phe Phe Asp Tyr Asp Tyr Gly Ala Pro Cys20 25 30 His Lys Phe Asp Val Lys Gln Ile Gly Ala Gln Leu Leu Pro Pro Leu35 40 45 Tyr Ser Leu Val Phe Ile Phe Gly Phe Val Gly Asn Met Leu Val Val50 55 60 Leu Ile Leu Ile Asn Cys Lys Lys Leu Lys Cys Leu Thr Asp Ile Tyr65 70 75 80 Leu Leu Asn Leu Ala Ile Ser Asp Leu Leu Phe Ile Ile Thr LeuPro 85 90 95 Leu Trp Ala His Ser Ala Ala Asn Glu Trp Val Phe Gly Asn AlaMet 100 105 110 Cys Lys Leu Phe Thr Gly Leu Tyr His Ile Gly Tyr Phe GlyGly Ile 115 120 125 Phe Phe Ile Ile Leu Leu Thr Ile Asp Arg Tyr Leu AlaIle Val His 130 135 140 Ala Val Phe Ala Leu Lys Ala Arg Thr Val Thr PheGly Val Val Thr 145 150 155 160 Ser Val Ile Thr Trp Leu Val Ala Val PheAla Ser Val Pro Gly Ile 165 170 175 Ile Phe Thr Lys Cys Gln Lys Glu AspSer Val Tyr Val Cys Gly Pro 180 185 190 Tyr Phe Pro Arg Gly Trp Asn AsnPhe His Thr Ile Met Arg Asn Ile 195 200 205 Leu Gly Leu Val Leu Pro LeuLeu Ile Met Val Ile Cys Tyr Ser Gly 210 215 220 Ile Leu Lys Thr Leu LeuArg Cys Arg Asn Glu Lys Lys Arg His Arg 225 230 235 240 Ala Val Arg ValIle Phe Thr Ile Met Ile Val Tyr Phe Leu Phe Trp 245 250 255 Thr Pro TyrAsn Ile Val Ile Leu Leu Asn Thr Phe Gln Glu Phe Phe 260 265 270 Gly LeuSer Asn Cys Glu Ser Thr Ser Gln Leu Asp Gln Ala Ile Gln 275 280 285 ValThr Glu Thr Leu Gly Met Thr His Cys Cys Ile Asn Pro Ile Ile 290 295 300Tyr Ala Phe Val Gly Glu Lys Phe Arg Arg Tyr Ile Ser Val Phe Phe 305 310315 320 Arg Lys His Ile Xaa Xaa Xaa Phe Cys Lys Gln Cys Pro Val Phe Tyr325 330 335 Arg Glu Thr Val Asp Gly Val Thr Ser Thr Asn Thr Pro Ser ThrGly 340 345 350 Glu Gln Glu Val Ser Ala Gly Leu 355 360 355 amino acidsamino acid single linear None 8 Met Thr Thr Ser Ile Asp Thr Val Glu ThrPhe Gly Thr Thr Ser Tyr 1 5 10 15 Tyr Asp Asp Val Gly Leu Leu Cys GluLys Ala Asp Thr Arg Ala Leu 20 25 30 Met Ala Gln Phe Val Pro Pro Leu TyrSer Leu Val Phe Thr Val Gly 35 40 45 Leu Ile Gly Asn Val Val Val Val MetIle Leu Ile Lys Tyr Arg Arg 50 55 60 Ile Arg Ile Met Thr Asn Ile Tyr LeuLeu Asn Leu Ala Ile Ser Asp 65 70 75 80 Leu Leu Phe Ile Val Thr Leu ProPhe Trp Thr His Tyr Val Arg Gly 85 90 95 His Asn Trp Val Phe Gly His GlyMet Cys Asn Leu Ile Ser Gly Phe 100 105 110 Tyr His Thr Gly Leu Tyr SerGlu Ile Phe Phe Ile Ile Leu Leu Thr 115 120 125 Ile Asp Arg Tyr Leu AlaIle Val His Ala Val Phe Ala Ile Arg Ala 130 135 140 Arg Thr Val Thr PheGly Val Ile Thr Ser Ile Val Thr Trp Gly Ile 145 150 155 160 Ala Val IleAla Ala Leu Pro Glu Phe Ile Phe Tyr Glu Thr Glu Glu 165 170 175 Leu PheGlu Glu Thr Ile Cys Ser Ala Leu Tyr Pro Glu Asp Thr Val 180 185 190 TyrSer Trp Arg His Phe His Thr Ile Arg Met Thr Ile Phe Cys Leu 195 200 205Val Leu Pro Leu Leu Val Met Ala Ile Cys Tyr Thr Gly Ile Ile Lys 210 215220 Thr Leu Leu Arg Cys Pro Xaa Xaa Xaa Lys Tyr Lys Ala Ile Arg Leu 225230 235 240 Ile Phe Val Ile Met Ala Val Phe Phe Ile Glu Trp Thr Pro TyrAsn 245 250 255 Val Ala Ile Leu Ile Ser Ser Tyr Gln Ser Leu Leu Phe GlyAsn Asn 260 265 270 Cys Glu Arg Ser Lys His Leu Asp Leu Val Met Ile ValThr Glu Val 275 280 285 Ile Ala Tyr Ser His Cys Cys Met Asn Glu Val IleTyr Ala Phe Val 290 295 300 Gly Glu Arg Phe Arg Lys Tyr Ile Arg His PhePhe His Arg His Leu 305 310 315 320 Leu Met His Leu Gly Arg Tyr Ile ProPhe Leu Pro Xaa Xaa Xaa Ile 325 330 335 Glu Arg Ile Ser Ser Val Ser ProSer Thr Ala Glu Pro Glu Ile Ser 340 345 350 Ile Val Phe 355 355 aminoacids amino acid single linear None 9 Met Glu Thr Pro Asn Thr Thr GluAsp Tyr Asp Thr Thr Thr Glu Phe 1 5 10 15 Asp Tyr Gly Asp Ala Thr ProCys Gln Lys Val Asn Glu Arg Ala Phe 20 25 30 Gly Ala Gln Leu Leu Pro ProLeu Tyr Ser Leu Val Phe Val Ile Gly 35 40 45 Leu Val Gly Asn Ile Leu ValVal Leu Val Leu Val Gln Tyr Lys Arg 50 55 60 Leu Lys Asn Met Thr Ser IleTyr Leu Leu Asn Leu Ala Ile Ser Asp 65 70 75 80 Leu Leu Phe Ile Phe ThrLeu Pro Phe Trp Ile Asp Tyr Lys Leu Lys 85 90 95 Asp Asp Trp Val Phe GlyAsp Ala Met Cys Lys Ile Ile Ser Gly Phe 100 105 110 Tyr Tyr Thr Gly LeuTyr Ser Glu Ile Phe Phe Ile Ile Leu Leu Thr 115 120 125 Ile Asp Arg TyrLeu Ala Ile Val His Ala Val Phe Ala Ile Arg Ala 130 135 140 Arg Thr ValThr Phe Gly Val Ile Thr Ser Ile Ile Ile Trp Ala Ile 145 150 155 160 AlaIle Ile Ala Ser Met Pro Gly Leu Tyr Phe Ser Lys Thr Gln Trp 165 170 175Glu Phe Thr His His Thr Cys Ser Leu His Phe Pro His Glu Ser Leu 180 185190 Arg Glu Trp Lys Leu Phe Gln Ala Leu Lys Leu Asn Leu Phe Gly Leu 195200 205 Val Leu Pro Leu Leu Val Met Ile Ile Cys Tyr Ile Gly Ile Ile Lys210 215 220 Ile Leu Leu Arg Arg Pro Asn Glu Lys Lys Ser Lys Ala Val ArgLeu 225 230 235 240 Ile Phe Val Ile Met Ile Ile Phe Phe Leu Phe Trp IlePro Tyr Asn 245 250 255 Leu Thr Ile Ile Ile Ser Val Phe Gln Asp Phe LeuPhe Thr His Glu 260 265 270 Cys Glu Gln Ser Arg His Leu Asp Leu Ala ValGln Val Thr Glu Val 275 280 285 Ile Ala Tyr Thr His Cys Cys Val Asn GluVal Ile Tyr Ala Phe Val 290 295 300 Gly Glu Arg Phe Arg Lys Tyr Ile ArgGln Leu Glu His Arg Arg Val 305 310 315 320 Ala Val His Leu Val Lys TrpLeu Pro Phe Leu Ser Val Asp Arg Ile 325 330 335 Glu Arg Val Ser Ser ThrSer Pro Ser Thr Gly Glu His Glu Ile Ser 340 345 350 Ala Gly Phe 355 360amino acids amino acid single linear None 10 Met Asn Pro Thr Asp Ile AlaAsp Thr Thr Leu Asp Glu Ser Ile Tyr 1 5 10 15 Ser Asn Tyr Tyr Leu TyrGlu Ser Ile Pro Lys Pro Cys Thr Lys Glu 20 25 30 Gly Ile Lys Ala Phe GlyGlu Leu Phe Leu Pro Pro Leu Tyr Ser Leu 35 40 45 Val Glu Val Phe Gly LeuIle Gly Asn Ser Val Val Val Leu Val Leu 50 55 60 Phe Lys Tyr Lys Arg IleArg Ser Met Thr Asp Val Tyr Leu Leu Asn 65 70 75 80 Leu Ala Ile Ser AspLeu Leu Phe Val Phe Ser Leu Pro Phe Trp Gly 85 90 95 Tyr Tyr Ala Ala AspGln Trp Val Phe Gly Leu Gly Ile Cys Lys Met 100 105 110 Ile Ser Trp MetTyr Leu Val Gly Phe Tyr Ser Gly Ile Phe Phe Val 115 120 125 Met Ile MetSer Ile Asp Arg Tyr Leu Ala Ile Val His Ala Val Glu 130 135 140 Xaa XaaXaa Ala Arg Thr Ile Ile Tyr Gly Val Ile Thr Ser Leu Ala 145 150 155 160Thr Trp Ser Val Ala Val Phe Ala Ser Leu Pro Gly Phe Ile Phe Ser 165 170175 Thr Cys Tyr Thr Glu Arg Asn His Thr Tyr Cys Lys Thr Lys Tyr Ser 180185 190 Leu Asn Ser Thr Thr Trp Lys Val Leu Ser Ser Leu Glu Ile Asn Ile195 200 205 Leu Gly Leu Val Ile Pro Leu Gly Ile Met Leu Phe Cys Tyr SerMet 210 215 220 Ile Ile Arg Thr Leu Gln His Cys Lys Asn Glu Lys Lys AsnLys Ala 225 230 235 240 Val Lys Met Ile Phe Ala Val Val Val Leu Phe LeuGly Phe Trp Thr 245 250 255 Pro Tyr Asn Ile Val Leu Phe Leu Glu Thr LeuVal Glu Leu Glu Val 260 265 270 Ile Gln Asp Cys Thr Phe Glu Arg Tyr LeuAsp Tyr Ala Ile Gln Ala 275 280 285 Thr Glu Thr Leu Ala Phe Val His CysCys Leu Asn Pro Ile Ile Tyr 290 295 300 Phe Phe Leu Gly Glu Lys Phe ArgLys Tyr Ile Ile Gln Leu Phe Lys 305 310 315 320 Xaa Xaa Xaa Gly Leu PheVal Ile Cys Gln Tyr Cys Gly Leu Leu Gln 325 330 335 Ile Tyr Ser Ala AspThr Pro Ser Ser Ser Tyr Thr Gln Ser Thr Met 340 345 350 Asp His Asp LeuHis Asp Ala Leu 355 360 54 amino acids amino acid single linear 11 ThrCys Ser Ser His Phe Pro Tyr Ser Gln Tyr Gln Phe Trp Lys Asn 1 5 10 15Phe Gln Thr Leu Lys Ile Val Ile Leu Gly Leu Val Leu Pro Leu Leu 20 25 30Val Met Val Ile Cys Tyr Ser Gly Ile Leu Lys Thr Leu Leu Arg Cys 35 40 45Arg Asn Glu Lys Lys Arg 50 147 base pairs nucleic acid single linear 12TTTCCATACA GTCAGTATCA ATTCTGGAAG AATTTCCAGA CATTAAAGAT AGTCATCTTG 60GGGCTGGTCC TGCCGCTGCT TGTCATGGTC ATCTGCTACT CGGGAATCCT AAAAACTCTG 120CTTCGGTGTC GAAATGAGAA GAAGAGG 147 34 amino acids amino acid singlelinear 13 Phe Pro Tyr Ile Lys Asp Ser His Leu Gly Ala Gly Pro Ala AlaAla 1 5 10 15 Cys His Gly His Leu Leu Leu Gly Asn Pro Lys Asn Ser AlaSer Val 20 25 30 Ser Lys 27 base pairs nucleic acid single linear 14TCGAGGATCC AAGATGGATT ATCAAGT 27 27 base pairs nucleic acid singlelinear 15 CTGATCTAGA GCCATGTGCA CAACTCT 27 20 base pairs nucleic acidsingle linear 16 CCTGGCTGTC GTCCATGCTG 20 27 base pairs nucleic acidsingle linear 17 CTGATCTAGA GCCATGTGCA CAACTCT 27

We claim:
 1. A protein comprising the amino acid sequence listed as SEQID NO:4.
 2. The protein of claim 1, consisting of the amino acidsequence listed as SEQ ID NO:4.
 3. A protein comprising the amino acidsequence listed as SEQ ID NO:6.
 4. The protein consisting of the aminoacid sequence listed as SEQ ID NO:6.